Axonal regeneration after injury or after inflammatory attacks or after neurodegenerative diseases within the mammalian central nervous system (CNS) is almost always impossible; the outcome depends on the balance between the intrinsic ability of the nerve fibers in the CNS to re-grow, and the inhibitory factors within the CNS, localized in the microenvironment of the lesion or damage site, which actively prevent the re-growth, and thus the regeneration of the injured fiber tracts.
It has been established that CNS myelin, generated by oligodendrocytes, and the lesional scar are the most relevant non-permissive structures for axonal growth in the early phase of an injury, by causing growth cone collapse and neurite growth inhibition in vitro as well as in vivo, thereby resulting in direct inhibition of axon regrowth. RGM proteins, major inhibitory factors on CNS myelin and scar tissue have been identified (Monnier et al., Nature 419: 392-395, 2002; Schwab et al., Arch. Neurol. 62: 1561-8, 2005a; Schwab et al. Eur. J. Neurosci. 21:1569-76, 2005 b; Hata et al. J. Cell Biol. 173:47-58, 2006; for reviews see: Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29, 2006; Yamashita et al. Curr. Opin. Neurobiol. 17: 29-34, 2007). RGM proteins are up-regulated at damage or lesion sites in humans dying from brain trauma or ischemic insult, (Schwab et al., Arch. Neurol. 62: 1561-8, 2005a) and are up-regulated at lesion sites in rats with spinal cord injury (Schwab et al. Eur. J. Neurosci. 21:1569-76, 2005 b; Hata et al. J. Cell Biol. 173:47-58, 2006 for review see: Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29, 2006; Yamashita et al. Curr. Opin. Neurobiol. 17: 29-34, 2007). In addition first data using clinical samples from Multiple sclerosis patients and healthy persons suggested that human RGM A is up-regulated in cerebrospinal fluid of patients suffering from MS (data not shown).
To evaluate the regeneration-promoting potential of a RGM A-specific polyclonal antibody, the antibodies were administered in a moderate-to-severe model of spinal cord injury, where approximately 60% of the spinal cord at thoracal level 9/10 was transected. The histological examination revealed that such a lesion severed all dorsal and lateral fibers of the corticospinal tract. The RGM A-specific polyclonal antibody given locally via pump for two weeks induced long-distance regeneration of injured nerve fibers (Hata et al., J. Cell Biol. 173:47-58, 2006).
Hundreds of nerve fibers extended past the lesion site and the longest fibers regenerated for more than 10 mm beyond the lesion, whereas no regenerating fibers were found distal to the lesion in control antibody-treated animals. The functional recovery of the anti-RGM A treated rats was significantly improved in comparison with control-antibody treated, spinally injured rats, thereby proving that RGM A is a potent neuroregeneration inhibitor and a valuable target for stimulating recovery in indications characterized by axon damage or nerve fiber injury (Hata et al., J. Cell Biol. 173:47-58, 2006; Kyoto et al. Brain Res. 1186: 74-86, 2007). In addition neutralising the RGM A protein with a function-blocking polyclonal antibody stimulated not only regrowth of damaged nerve fibers in the spinally injured rats but enhanced their synapse formation thereby enabling the reformation or restoration damaged neuronal circuits (Kyoto et al. Brain Res. 1186: 74-86, 2007).
The rgm gene family encompasses three different genes, two of them, rgm a and b, are expressed in the mammalian CNS originating RGM A and RGM B proteins, whereas the third member, rgm c, is expressed in the periphery (Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29, 2006), where RGM C plays an important role in iron metabolism. In vitro, RGM A inhibits neurite outgrowth by binding to Neogenin, which has been identified as an RGM receptor (Rajagopalan et al. Nat Cell Biol.: 6(8), 756-62, 2004). Neogenin had first been described as a netrin-binding protein (Keino-Masu et al. Cell, 87(2):175-85, 1996). This is an important finding because binding of Netrin-1 to Neogenin or to its closely related receptor DCC (deleted in colorectal cancer) has been reported to stimulate rather than to inhibit neurite growth (Braisted et al. J. Neurosci. 20: 5792-801, 2000). Blocking RGM A therefore releases the RGM-mediated growth inhibition by enabling Neogenin to bind its neurite growth-stimulating ligand Netrin. Based on these observations, neutralizing RGM A can be assumed to be superior to neutralizing neogenin in models of human spinal cord injury. Besides binding of RGM A to Neogenin and inducing neurite growth inhibition, the binding of RGM A or B to the bone morphogenetic proteins BMP-2 and BMP-4 could represent another obstacle to successful neuroregeneration and functional recovery (Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29, 2006).
There is a need in the art for improved antibodies capable of binding RGM A, preferably a monoclonal antibody that blocks RGM A and prevents the interaction between RGM A and its receptor and/or binding proteins, i.e. Neogenin and BMP-2, BMP-4.
The present application provides (a) the generation of a neutralizing monoclonal antibody against RGM A, which selectively inhibits binding of RGM A to its receptor Neogenin and to bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4), and (b) the generation of a neutralizing monoclonal antibody against RGM A, which selectively inhibits binding of RGM A to bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4). The neutralizing monoclonal antibodies of the present invention are expected to stimulate regrowth of injured or damaged nerve fibers and formation of functional synapses of regenerating nerve fibers since one of the neutralizing monoclonal antibodies of the present invention appears to transform the inhibitory nature of RGM A in a condition in which neuronal cells prefer to migrate and grow on an RGM A substrate, and not on a permissive substrate like Collagen I. In addition this antibody is able to induce long-distance regeneration in an in vivo rat model of optic nerve injury and it also enhances remyelination of lesioned and regenerating nerve fibers.
Accordingly, the neutralizing monoclonal antibodies of the present invention are expected to promote neuronal regeneration and regrowth of damaged or broken neuronal connections in the injured and inflamed human CNS, for example in multiple sclerosis, after acute spinal cord injury, brain trauma, or in neurodegenerative diseases such as for example, Huntington's chorea, Parkinson's disease, Alzheimer's disease.